The mammalian tooth germ provides a powerful system for studying the epithelial-mesenchymal tissue interactions that underlie organogenesis. In the prior grant, we investigated the function of the Msx1 homeobox gene in mouse molar tooth development, and identified components of the genetic pathway involved. We found that BMP4 effects a highly specific and nearly complete rescue of Msx1 mutant molar tooth development, which otherwise arrests at the bud stage. Thus, BMP4 is a key downstream effector of Msx1 function in the dental mesenchyme during odontogenesis. In addition, Msx1 is required to integrate the mesenchymal response to different epithelially-derived inductive signals such as BMPs and FGFs that act on the dental mesenchyme. Msx1 also executes a later function in odontoblast survival, and its homolog Msx2 plays an essential role in enamel organ development. Nonetheless, the precise molecular function of Msx1 during early tooth development remains unsolved. For example, although candidates exist, there are no proven direct molecular regulators of Msx1 expression in the dental mesenchyme, nor are there direct downstream targets in the dental mesenchyme that are regulated by Msx 1. This competing continuation will address this gap in our knowledge. Tooth development in both Msx1 and Pax9 knockout mice arrests at the bud-stage, and both genes are expressed in a hierarchical manner in dental mesenchyme. These data suggest a close molecular relationship between Pax9, Msx1 and other components of the early odontogenic hierarchy. This competing renewal proposes three Specific Aims to clarify this relationship and to identify new regulators that act at the bud stage of tooth development. First, we will use transcriptional profiling and bioinformatic approaches to identify genes whose expression is deregulated in Msx1 and Pax9 mutant dental mesenchyme. Second, we will use the information from Aim I along with experimental approaches to identify transcription factors that directly regulate Msx1 expression in the dental mesenchyme. Third, we will clarify the mechanism by which Msx1 acts in bud stage dental mesenchyme by identifying Msx1-interacting proteins and by testing their ability to co-regulate presumptive Msx1 target genes, some inferred from Aim 1. Collectively, these experiments should define the early odontogenic regulatory hierarchy in both genetic and molecular terms, clarify the relationship between two key regulators of early odontogenesis, Pax9 and Msx1, am enhance our general understanding of odontogenesis.